DNA Methyltransferase 32 nM (IC50) DNMT1 1.5 nM (Kd) DNMT3A 92 nM (IC50) DNMT3B 1000 nM (IC50) G9a 16 nM (IC50)

CM-579 has a Kd of 1.5 nM for DNMT1. With an IC50 of 92 nM for DNMT3A and 1000 nM for DNMT3B, respectively, CM-579 likewise inhibits these enzymes[1].

CM-272 (CM-579 analog) shows anti-leukaemic effects in vivo. [1]
ALL-derived CEMO cells (10 × 106) were injected i.v. in immunodeficient Rag2−/−γc−/− mice, which were treated with 2.5 mg kg−1 of CM-272 administered daily, starting 3 days after injection and continued during 28 days. Control animals received saline solution under the same protocol. CM-272 therapy induced a statistically significant increase in overall survival (OS) in mice in comparison with control animals (median OS; 92±5.7 days versus 55±10.5 days; P=0.0009) (Fig. 4a). Global H3K9me2 and 5mC levels were measured in the extract from total liver. Tumour infiltration was analysed in liver homogenates by flow cytometry (FACS) analysis and showed an infiltration of 60–80% of human cells (hCD45+). Both marks were reduced in leukaemic cells obtained from animals after 1 week of treatment (Supplementary Fig. 10f). No significant weight loss was observed in treated animals (Supplementary Fig. 10g). We obtained similar results in a second in vivo replicate with CEMO-1 cells (Supplementary Fig. 11a). To analyse the dose-dependent efficacy of CM-272 in vivo, we repeated the same study administering 1 mg kg−1 of CM-272 (Supplementary Fig. 12). We did not observe differences in the body weight of the animals (Supplementary Fig. 12a) nor significant changes in haematological parameters (Supplementary Fig. 12b) between mice treated with 1 mg kg−1 or 2.5 mg kg−1 of CM-272 and the control group. As expected, CM-272 plasma concentration was greater in the mice group treated with 2.5 mg kg−1 of CM-272 (Supplementary Fig. 12c). However, treatment with 1 mg kg−1 of CM-272 was not able to prolong survival of the mice unlike what was observed when the 2.5 mg kg−1 of CM-272 was used (Supplementary Fig. 12d). These results demonstrate dose-dependent efficacy of CM-272 and that a dose of 2.5 mg kg−1 of CM-272 is adequate to demonstrate the positive anti-tumour efficacy. In a second xenogeneic model, 10 × 106 of AML-derived MV4-11 cells were injected i.v. in Rag2−/−γc−/− mice, and 14 days latter animals were treated with 2.5 mg kg−1 of CM-272 for 28 days. As in ALL cells, CM-272 therapy prolonged OS in mice (median OS for treated versus untreated mice, 78±12 days versus 57±0.9 days; P=0.0005) (Fig. 4b). We obtained similar results in a second in vivo replicate with MV4-11 cells (Supplementary Fig. 11b), without any sign of toxicity (Supplementary Fig. 10h). Finally, 2.5 × 106 cells from the OCI-Ly10 activated B-cell DLBCL cell line were similarly i.v. injected into Rag2−/−γc−/− mice. Treatment with CM-272 at the same dose during 8 weeks also prolonged OS of treated mice in comparison to control animals (median OS; 59±8 days versus 49±6 days; P=0.010) (Fig. 4c). We obtained similar results in a second in vivo replicate with OCI-Ly10 cells (Supplementary Fig. 11c), without any sign of toxicity (Supplementary Fig. 10i). Although the effect on lymphoma cells was statistically significant, the effect was less robust than in the case of AML and ALL cells. These results show that CM-272 exerts a potent anti-tumour activity in vivo against different types of haematological malignancies by inhibiting the methyltransferase activity of both G9a/GLP and DNMTs. In addition to the information described above, minimal promiscuity versus other SAM-dependent epigenetic enzymes (Supplementary Tables 4a and 4b), further off-target selectivity profiling against other drug targets in cancer (a panel of 97 kinases, Supplementary Tables 12–14) confirmed G9a (and GLP) and DNMTs as primary main targets for CM-272.

G9a and DNMT1 enzyme activity assays [1]
G9a and DNMT1 activities were measured using a time-resolved fluorescence energy transfer (TR-FRET). For G9a, TR-FRET is observed when biotinylated histone monomethyl-H3K9 peptide is incubated with cryptate-labelled anti-dimethyl-histone H3K9 antibody and streptavidin XL665 after enzymatic reaction of G9a. For DNMT1, TR-FRET is observed when antibody specific to S-adenosylhomocysteine labelled with Lumi4-Tb (donor) is incubated with d2-labelled S-adenosylhomocysteine (acceptor), using the EPIgeneous methyltransferase assay. Details are provided in Supplementary Information. The radioligand binding assay against G9a, DNMT1 and GLP was performed by Reaction Biology Corporation (http://www.reactionbiology.com).

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Epigenetics selectivity panel[1]
Selectivity of CM-272 and CM-579 against 37 epigenetic enzyme targets including Bromodomain-containing enzymes (ATAD2A, ATAD2B, BAZ2B, BRD1, BRD2(BD1+BD2), BRD4(BD1+BD2), BRDT(BD1), CREBBP, TRIM24, TAF1), Histone methyltransferases (EZH1, EZH2, GLP, PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT8, SETD2, SET7/9, SUV39H1, SUV39H2 and MLL-WARD), DNA methyltransferases (DNMT3A and DNMT3B) and histone demethylase (JMJD2A, JMJD2B, JMJD2C, JMJD2D, JMJD2E, JMJD3, JMJD1A, LSD1, Jarid1A, Jarid1B and Jarid1C) was performed by BPS Bioscience (http://www.bpsbioscience.com/index.ph).

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HDAC1 and HDAC6 enzyme activity assays[1]
HDAC1 and HDAC6 enzyme activities were measured with a specific fluorescence-labelled substrate, containing an acetylated lysine side chain, after its deacetylation by HDACs. Details are provided in Supplementary Information.

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Kinase selectivity profiling[1]
The selectivity profiling of CM-272 against a selected panel of 97 kinases distributed through the kinome (out of which 90 are non-mutant kinases) was performed at DiscoverRx (http://www.discoverx.com/home) using the KINOMEscan screening platform at a test concentration of 10 μM.

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Direct binding analysis[1]
MicroScale Thermophoresis (MST) was performed to quantify biomolecular interactions between CM-579 and DNMT1 (full length). The MST analysis was performed using the Monolith NT.115 instrument.

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ADME profiling[1]
The following ADME studies: CYP inhibition on five human cytochrome P450s (1A2, 2C9, 2C19, 2D6 and 3A4 at 10 μM) in human liver microsomes, plasma protein binding, kinetic solubility, Pampa permeability and human and mouse liver microsomal stability were performed by Wuxi (http://www.wuxi.com/).

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hERG blockade assay[1]
The effect of the compound on hERG potassium channels was determined using the PredictorTM hERG fluorescence polarization commercial assay kit.

Cell Proliferation Assay[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4- 11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Inhibited cell proliferation in a dose- and time-dependent manner.

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Cell Cycle Analysis [1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells) ; 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 24 hrs (hours)
Experimental Results: Blocked cell cycle progression.

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Apoptosis Analysis[1]
Cell Types: CEMO-1, MV4-11 and OCI-Ly10 cell lines
Tested Concentrations: 125 nM, 250 nM, 500 nM (CEMO-1 cells); 135 nM, 270 nM, 540 nM (MV4-11 cells); 100 nM, 400 nM, 1000 nM (OCI-Ly10 cells)
Incubation Duration: 12 hrs (hours), 24 hrs (hours), 48 hrs (hours) and 72 hrs (hours)
Experimental Results: Induced apoptosis in ALL, AML and DLBCL cell lines in a dose- and time-dependent manner.

Animal/Disease Models: Female BALB/Ca-Rag2−/−γc−/− mice (6–8weeks old) with CEMO-1 cells[1]
Doses: 2.5 mg/kg
Route of Administration: intravenous (iv) injection; daily; for 28 days
Experimental Results: Induced a statistically significant increase in overall survival (OS) in mice.

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PK study of CM-272 and CM-579 in plasma samples [1]
CM-272 and CM-579 were measured in plasma samples using a Xevo-TQ MS triple quadropole mass spectrometer with an electrospray ionization (ESI) source and an Acquity UPLC. CM-272 and CM-579 solutions were prepared by dissolving the solid in saline. A drug dosage of 1 mg kg−1 or 2.5 mg kg−1 (CM-272) or 1 mg kg−1 (CM-579 ) was administered as a single intravenous injection. Blood was collected at predetermined times over 24 h post injection (0.25, 2, 4, 6, 8 and 24 h for CM-272) and (0.25, 1, 2, 4 and 8 h for CM-579). Chromatographic separation was performed by gradient elution at 0.6 ml min−1 using an Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm particle size). The PK parameters were obtained by fitting the blood concentration-time data to a non-compartmental model with the WinNonlin software. Details are provided in Supplementary Information.

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In vivo experiments[1]
The human ALL CEMO-1 (control group with saline solution n=6; treated group with CM-272, n=6), AML MV4-11 (control group with saline solution n=8; treated group with CM-272 n=8) and DLBCL OCI-Ly10 (control group with saline solution n=6; treated group with CM-272 n=6) xenograft mice models were generated by i.v. injection of cells diluted in 100 μl of saline solution in the tail vein of a 6–8-week-old female BALB/cA−Rag2−/−γc−/− mice as described in Supplementary Information. CM-272 administration was detailed in Supplementary Information. Statistical results were calculated using the statistical software medcalc.

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CM-272 toxicity assay: haematological and liver parameters[1]
After treating Rag2−/−γc−/− mice with daily i.v. 2.5 mg kg−1 of CM-272 during 4 weeks, followed by a 7 days washout period, haematological and liver parameters were measured as described in Supplementary Information.

Before performing PK studies, researchers identified the maximum tolerated dose (MTD) for CM-272, which it was at 2.5 mg kg−1 (intravenous, i.v.); however, for CM-579 we could not administer a dose higher than 1 mg kg−1 (i.v.). PK studies that researchers performed using the MTD dose (Supplementary Tables 9–11), showed clearance levels for CM-579 (5.7 l h−1 kg−1) and CM-272 (0.91 l h−1 kg−1).

Before evaluating the in vivo efficacy of our dual inhibitors, researchers examined the therapeutic window achieved by these two molecules and their pharmacokinetic (PK) parameters. Thus, we studied toxicity of CM-272 and CM-579 using the non-tumoural hepatic cell line THLE-2 (LC50s were 1.78 and 1.30 μM) as well as peripheral blood mononuclear cells (PBMCs) obtained from healthy donors (LC50s were 1.90 and 7.39 μM) (Supplementary Table 2) and compared with the in vitro activity against tumour cell lines (Supplementary Table 3). Researchers detected that CM-272 and CM-579 showed an acceptable therapeutic window (around 1 log units). Before performing PK studies, we identified the maximum tolerated dose (MTD) for CM-272, which it was at 2.5 mg kg−1 (intravenous, i.v.); however, for CM-579 researchers could not administer a dose higher than 1 mg kg−1 (i.v.). PK studies that we performed using the MTD dose (Supplementary Tables 9–11), showed clearance levels for CM-579 (5.7 l h−1 kg−1) and CM-272 (0.91 l h−1 kg−1). [1]

[1]. Discovery of first-in-class reversible dual small molecule inhibitors against G9a and DNMTs in hematological malignancies. Nat Commun. 2017 May 26;8:15424.

CM-272 is a member of the class of aminoquinolines that is is quinoline substituted by 5-methylfuran-2-yl, (1-methylpiperidin-4-yl)amino, methoxy, and 3-(pyrrolidin-1-yl)propoxy groups at positions 2, 4, 6 and 7, respectively. It is a dual G9a/DNA methyltransferases inhibitor with antitumor activity. It inhibits G9a, DNMT1, DNMT3A, DNMT3B and GLP (IC50 = 8 nM, 382 nM, 85 nM, 1200 nM and 2 nM, respectively). It has a role as an apoptosis inducer, a ferroptosis inducer, an antineoplastic agent, an EC 2.1.1.43 (enhancer of zeste homolog 2) inhibitor and an EC 2.1.1.37 [DNA (cytosine-5-)-methyltransferase] inhibitor. It is a N-alkylpyrrolidine, a member of furans, an aminoquinoline, an aromatic ether, a member of piperidines, a tertiary amino compound, a secondary amino compound and a diether.

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The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.[1]

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The results of the transcriptomic analysis performed after treatment with CM-272 consistently suggest a tumour interferon type 1 response with expression of ISGs and induction of immunogenic cell death (ICD) in AML, ALL and DLBCL cells, pointing to a common mechanism of anti-tumour effect. Despite the fact that ICD had not been described as a mechanism of action for epigenetic drugs, these results might have been at least partially predicted, because recent studies have shown that expression of ISGs is epigenetically regulated by H3K9me2 (ref. 30), supporting the role of G9a inhibition in the activation of type I interferon responses and ICD. Thus, we speculate that the use of immunocompromised mice unable to develop anti-tumour immune responses may have underestimated the efficacy of CM-272 against tumour cells, prompting the evaluation of immune-competent models to explore the full potential therapeutic effects of our compounds33. On the basis of recent studies demonstrating that type I interferon responses contribute to the efficacy of chemotherapeutic agents32, the use of CM-272 in combination with such drugs and/or with immune modulators such as checkpoint inhibitors might also represent an attractive therapeutic strategy.
In summary, CM-272 is a potent novel first-in-class dual reversible inhibitor of G9a (GLP) and DNMTs that prolongs survival in in vivo models of haematological malignancies by at least in part inducing immunogenic cell death. These compounds represent a novel approach for targeting cancer safely and efficiently, paving the way for treating a broad series of human tumours with poor prognosis.[1]

C29H43CL3N4O3

602.04

600.2400

2448471-08-5

CM-579;1846570-40-8

Light yellow to yellow solid powder

XKSDREMVFCQUPC-UHFFFAOYSA-N

InChI=1S/C29H40N4O3.3ClH/c1-21-7-8-27(36-21)26-18-24(30-20-22-9-14-32(2)15-10-22)23-17-28(34-3)29(19-25(23)31-26)35-16-6-13-33-11-4-5-12-33;;;/h7-8,17-19,22H,4-6,9-16,20H2,1-3H3,(H,30,31);3*1H

6-methoxy-2-(5-methylfuran-2-yl)-N-[(1-methylpiperidin-4-yl)methyl]-7-(3-pyrrolidin-1-ylpropoxy)quinolin-4-amine;trihydrochloride

CM-579 trihydrochloride; CM579 triHCl; CM 579 3HCl; 2448471-08-5;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~37.5 mg/mL (~62.29 mM)
DMSO :~33.33 mg/mL (~55.36 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 16.67 mg/mL (27.69 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)